Recovery of vitamin b12 from liver



Patented Sept. 1952 moovnar F VITAMIN as mom uvan.

Norman G. Brink, Princeton, and Thomas a. Wood. Murray'Hill, N. 1., assignors to ck & Co.. Inc., Railway, N. J., a corpora New Jersey I of;

No Drawing. Application April 3, 1948,

Serial No. 18,854

This invention relates generally to the prep-' aration of therapeutic materials and, more particularly. to the preparation of vitamin Bu from liver.

Vitamin B12 is a water soluble redv colored crystalline compound containing carbon,- hydrogen, oxygen, nitrogen, phosphorus, and cobalt. This vitamin derived from liver is valuable in the treatment of certain types of human anemias. It

is a potent growth factor for the micro-organism Lactobacillus lactis Dorner. r

It is an obiect'of this invention to prepare vitamin B1: in pure form from commercial liver preparations.

We have found that it is possible to subject commercial liver preparations to a purification process whereby vitamin B1: is recovered possessing high physiological activity, particularly suitable for the clinical treatment of human anemia.

We have discovered that we can obtain vitamin B1: in a concentrated and purified form from commercial liver preparations by a chromatographic 1 Claim. (01. 167-81) 2 and the ethanol-insoluble solids discarded. The

ethanol solution is concentrated to dryness. and

after dissolving the residue in methanol. acetone or ether is added to effect precipitation. The supernatant liquid is then discarded. the precipitate dissolved in water and the solution treated with acetone. The resulting precipitate is dissolved in methanol and several volumes of acetone are added. The supernatant liquid is discarded and the residue crystallized by solution in water and careful addition of acetone to turbidity. The resulting crystals are washed with acetone and recrystallized from water-acetone mixtures.

" Alternatively, the vitamin B1: concentrates. after the solvent-precipitation steps may be further purified by counter-current distribution between water and a mixture of 75% toluene-% o-cresol, followed by crystallization of the best fractions.

If desired, the concentrates obtained by treat ment with activated charcoal may be dissolved in'a lower aliphatic alcohol and the resulting altechnique. The purification andconcentration is carried out under closely controlled conditions whereby the possibility of destruction or loss of the activity of the vitamin is reduced to a minimum.

According to the process of our inventionwe use as starting material-aliver fraction of at least 1000 units per mg. which is prepared from commercially available preparations such as the 70% ethanol soluble fraction from an aqeous liver extract by the method such as that shown by Dakin A and West in Jour. Biol. Chem. 109, 489 (1935).

The solid material thus obtained is chromatographed in aqueous solution on a column containing charcoal and the active material eluted with an organic solvent such as 50% ethanol. Other eluants such as 50% acetone, butanol-saturated water, benzyl alcohol-saturated water, and glacial acetic acid may also used.

Alternatively according to our preferred process. the alcoholic solution prepared by extraction of the solid material with a lower aliphatic alcohol is chromatographed on a column containing activated alumina and the active material eluted with methanol or aqueous methanol.

The eluates are collected in small fractions. The potency of the fractions is determined by microbiological assay by Lactobacilluslactis Dorner. The fractions of high potency are either dried, from the frozen state, or concentrated to a small volume.

Chromatographed material either solid or. concentrated eluate such as that obtained from-the alumina chromatogram is extracted with ethanol,

'coholio solution may be chromatographed on a column ofactivated alumina.

The activity of the products is determined microbiologically by means of a growth response with Lactobacillus lactis Dorner as the test organism. .An arbitrarily selected liver concentrate was accorded a value of 1000 units per mg.

A briefdescription of the assay method which we employed is given below.

Lactobacillus lactis Domer has been reported to require'two growth factors, T. J. and L. L. D. Medium modifications incorporated in the formula described below, have eliminated the requirement for the T. J. factor, and the assay as v presented is specific for L. L. D. This microorganism shows L. L. D. factor response to vitamin B12. Pure crystalline vitamin B12 is used as the assay standard and all unknowns evaluated in terms of microbiological growth-promoting activity, equivalent to the microbiological activity 'of vitamin Biz.

1% Difco yeast extract 0.02% tomato juice serum 1% anhydrous dextrose 1.5% agar The medium used for the inoculum consists of the basal or assay medium to which 1 unit per cc. of vitamin B12 is added. The inoculum cells are aeoases washed with sterile distilled water and diluted to forms. suspension which reads between 90% and 95 -light transmission on the Evelyn photometer with a 520 mu filter.

The composition of the assay medium, double strength. is listed below. Ingredients may be conveniently dispensed from stock solutions:

DL isoleucine mg 200 DL alpha-alanine mg 200 D1. aspartic acid ...mg 200 DL valine mg 200 DL methionine m 200 DL glutamic acid mg 200 DL threoni mg 200 DL serine m 200 DL phenylalanine mg' 200 DL leucinemg 200' L histidine. mg 200 DL tryptophane ...mg.; 400

L arginine mg 200 L lysine I -mg 100 Amlnoacetic acid mg 200 L cystine m 200 DL norleucine mg 200 L tyrosine mg 200 Dextrose gm Sodium acetate gm 6 Fumaric acid gm 0.5 Sodium ethyloxalacetate gm 0.5 Riboflavin cg 200 Calcium pantothenate; mcg 200 'I'hiamin HG! mcg.. 200 Nicotinic acid mcg 200 Pyridoxamine mm: 400 Para-aminobenzoic acid mcg 40 Biotin meg..- 0.4 lhSOc'lHaO mg 200 NaCl mg" 10 FeSOa'IHaO mg 10 MnSOaAHgO mg 10 mo; mg 500 KHzPO4 500' Mile acid mcg 2 Casein hydrolyzate.. gm 1.0 Water to 500 cc.

The basal mediumis prepared by combining the amino acids, then adding dextrose, sodium acetate. fumaric acid, heating to dissolve, and immediately readjusting to pH 7. The sodium ethyloxalacetate and vitamins are then added,

dissolved, and the solutions again adjusted to pH 7. Finally, the salts, folic acid and casein hydrolyzates are added, dissolved, and the pH adjusted A water solution or suspension of the sample to be assayed is diluted so that the solution contains about 0.2 L. L. D. type units per cc. which is then added to the-assay tubes in 0.5, 1.0, 1.5, 2.0, and 2.5 cc. amounts. All of the tubes are then adjusted to a volume of 2.5 cc. with water, 2.5 cc. of the basal medium added. and the tubes are iinalLv plugged and sterilized by heating at 120 C. for 13 minutes. After cooling to room temperature, the tubes are inoculated with one drop of the standardized suspension of L. lactis and then incubated at 37 C. for 40 hours. After incubation, the tubes are titrated directly with 0.05 N sodium hydroxide to the blue green endpoint of bromthymol blue.

The activity of the sample may then be determined irom the standard curve by the amount of sodium hydroxide required.

The standard curve is prepared with crystalline B12 contains 11x10 units. A stock solution is diluted and 0.0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0 and 2.0 units added to duplicate series of tubes. Water is added to adjust the volume to 2.5 cc. The procedure described above for the samples under assay is followed with the standard. Typical titration values for the standard series listed above are 1.5, 2.0, 2.7, 4.3, 5.7, 6.6, 7.5, 79,-8.0 and 8.4 respectively, in terms of milliliters of 0.05 N sodium hydroxide required to neutralize the acid produced per culture of L. lactis.

The following examples illustrate a method of carrying out the present invention, but it is to be understood that these examples are given by way of illustration and not of limitation.

\ EXAMPLE 1 One gram of a concentrate having a micro biological potency of 3200 units per mg. was dissolved in 10 ml. of water and chromatographed on a column containing 10 g. of activated charcoal, preferably acid-washed. The column was washed thoroughly with water and theactive material then eluted with 50% ethanol. The eluate was collected as a series of fractions of about 10 ml. each. The fractions of highest potency, as measured by microbiological assay, were combined and dried from the frozen state. The dried material (115 mg.) had a microbiological potency of about 10,000 units per mg., representing a yield of about 50%.

EXAMPLE 2 A 6,000 unit/mg. concentrate (14.8 g.) containing a total of 87 million units was extracted three times by stirring for about 30 min. with 200 ml. of

methanol. The combined extracts were passed through a column about 7.5 cm. in diameter containing 600 g. of activated alumina. When the last of this solution had enteerd the alumina, methanol was added and allowed to percolate through the column for development of the chromatogram. During this operation, a pink band moveddown and through the column, leaving essentially all of the brown-yellow color of the original solution at the top of the column. The eiiiuent was collected in a series of fractions, of which those showing the most pronounced microbiological activity and/or the most pronounced pink color were selected and combined for further operations. This solution. which contained by microbiological assay about 65 million units, was evaporated in vacuo at a temperature below 25 C. to a thin syrup. During the evaporation, the pH of the solution, which tends to rise, was maintained at about 6 to 7 by the addition of dilute (2.5 N) hydrochloric acid as necessary. The residue was finally dried at low pressure (1 mm. mercury) at room temperature.

EXAMPLE 3 245 mg. of red residue from the alumina chromatogram was extracted with 15 ml. of absolute ethanol, giving a 34 mg. colorless insoluble residue and a red solution. The red solution atforded 218 mg. of a red oil. The red oil was ossolved in 0.5 ml. of methanol and 25 ml. of acetone was addel, when a red precipitate and yellow supernatant were obtained.

This precipitate (31 mg.) gave an assay value of 1 to 7 million units/mg, and had an absorption maximum at 3600 A. E%=26.9. It was reprecipitated from water (0.75 ml.) by acetone (13 ml.). The resulting red oil, 12.8 mg., was again vitamin B12. One milligram of pure crystalline precipitated from methanol solution with acefrom tubes numbered 2 through 5 (tube repretone, without any further weight loss. The prodmaximum at 8600 5.,

- After. two days the mother liquor was removed and the crystals were washed with acetone and dried, yielding 2.0 mg. An additional 1.4 ms. of crystals was obtained from the mother liquor.

' EXAMPLE 4 1 A 6.0 mg. portion of vitamin B1: concentrate of Example 3 with an activity of about 1 to 2X10 u./mg. was iractionated in a ten plate countercurrent distribution with water and a 75% toluene 25% o-cresol mixture mutually saturated as the'two immiscible phases. Two milliliters of each phase was used per tube, and the loweriaqueous) phases were transferred. At the completion of the distribution. the products were displaced to the aqueous phases by addition of 8 mi. of chloroform per tube. The aqueous phases crystalline vitamin B12 so obtained weighed about A 3.2 mg. portion of once-crystallized vitamin B1: from liver was dissolved in methanol. filtered, and the filtrate concentrated to dryness in vacuo.

. 6 In the L. L. D. assay. a sample of recrystallized vitamin 31: showed a value (corrected for weight loss in drying) of 11.2)(10 -u./mg.i-0.0X10'. u./mg. This corresponds to a half-maximal growth of L. lactis Dorner in the presence of 0.000013 'y/ml. of culture medium under the conditions used.

0n the micro hot-stage, the recrystallized vitamin B1 darkened to black at about 210-220", but did not liqueiy below 300.

Vitamin B1: concentrates obtained in accordance with this invention possess high therapeutic value in the treatment of certain types of human anemia such as Addisonian pernicious anemia and the like. thus the crystalline product has produced a positive hematological response in three The residue was dissolved in 0.15 ml. of water and acetone (ca. 1 ml.) was added until the solution became turbid; the solution. was seeded and allowed to stand overnight at room temperature. The crystals which had been deposited were separated from the mother liquor, washed with mpatients following single intramuscular injections of 150, 6, and 3 respectively. Four patients receiving. single injections of the amorphous concentrates containing 20 000 to 40.000 L. L. D. units gave strong or maximal hematological responses. One mg. of the crystalline material is equivalent to about 11x10 L. L. D. units and one 1 to about 11,000 units. The abbreviations used in Tables I v and II have the following significance:

RBCxl0=red blood cellsx 10 per cu. mm. Hgb. gm.=hemoglobin gm.( 100 cc. Retics, percent=reticulocytes, percent.

Hot; percentrL-hematocrit volume percent of cells.

WBCx10'=white blood pellsX 10 per cu. mm.

-' The patient went home on the th day andhas received no treatment aside from that-retone, and dried. The yield was about 2 mg. corded.

' Table II nitcarsumznn vrramm B,

owe a o D" Day 0 s 10 2a 0 0 14 i 0 s 0 is nncxw. 1.5 as 3.4 1.0 2.0 1.4 2. Hgb. 0a 1.0 0.0 1.2; 10.0 as 0. Retics.,percent 00 21.0 2.0 0.5 2.0 200 0.1 2.8 102 4.0 2. Hot. 110 2110 20.0 11.0 s1 11 a1 wsoxio 0.0 2110 0.0 20 as 4.2 s.1 Vitamin B127 150 6 3 1.; 1.. 1). Units 1.0)(10' 0.6)(10 3.3Xl0 110x10 The recrystallized vitamin 312 had, after dry Vitamin B12 is also capable of increasing the ing in vacuo at for two hours, an ultram growth rate of chicks on a basal diet containing The needle-like. birefringent crystals (sign of elongation, positive) have. after drying, the inall k'nown'nutrients. For. example. in one experiment a level of 0.000003% of the crystalline vitamin in the diet produced a response such that the average weight of the chicks after a s5 16-day feeding period was 124 g., compared to an average weight of g. for the control group whose ration was not supplemented with vitamin Modifications may be made in carrying out the 10 present'invention without departing from the spirit and scope thereof, and the invention is to be limited only .by the appended claim.

We claim:

The process for recovering vitamin B1: from a (1 08801 r fr ti n: 1 6:}. L 52; and'y. L 76 liver concentrate having an activity for Lactoa aeoaass 7 8 bacillus lactic Dorner in excess of 1,000 units per Number Name Date milligram which comprises passing a methanol 2.175.014 Booher Oct. 3. 1939 solution of said concentrate over a column 0! ac- 2,202,307 Booher May 28, 1940 tivated alumina. to adsorb said vitamin Bu. eluting said column with methanol, collecting 5' FOREIGN PATENTS the methanol eluate in successive fractions, re- Number Country t taining those fractions which have relatively high 250,230 Great Britain June 29, 1927 activity for Lactobacillus lactic Dorner and (115- 473,064 Great Britain Oct. 5, 1937 carding the other fractions, recovering a concen- 59 5 Great m m 1' 7 trate containing vitamin B1: in an enhanced state 10 of purity from the retained fractions or the 1 OTHER REFERENCES methanol eluate, purifying said vitamin B1: con- Fantes: Proceedings of the Royal Society centrate by counter-current distribution between (1950). pages 592 and 598. 167-81 Bu. water and a mixture of ortho-cresol and toluene, Emery: Proceedings of the Biochemical Sociedisplacing the active material to the water phase 15 ty. volume 40, (1946), page iv. 167-312.

by. adding chloroform, and precipitating the vita- 131111.312 in crystalline form by treating the aqueous solution with acetone. r NORMAN G. BRINK.

THOMAS R. WOOD.

REFERENCES CITED The following references are of record in the file of this patent: a

om'mn sums PATENTS Number Name Date Laland Oct. 25. was 

